T cell immunity plays a major role in determining the outcome of infection. Chronic infections are often distinguished by T cell responses that are not able to fully eliminate the pathogen. The mechanisms that explain this failure of T cell effector responses are only beginning to be understood. During the current funding period, we have used the LCMV model to investigate how the PD-1 pathway regulates T cell responses during acute and chronic infection. We have discovered that the PD-1+ stem-like population is responsible for the dramatic expansion of CD8+ T cells after PD-1 pathway. In addition, we identified roles of the PD-1 pathway in regulating CD8 and humoral responses during acute viral infection. However, the mechanisms of PD-1 signaling and its blockade are still not well understood. A deeper understanding of PD-1 signaling is needed to determine how to improve anti-viral immunity, while minimizing autoimmunity and immunopathology. To address this issue, we worked with Core C to generate novel PD-1 signaling domain mutant mice. We have found that both the PD-1 ITIM and ITSM motifs mediate PD-1 signaling in vivo. Notably, our data suggest the possibility to dissociate beneficial effects of PD-1 pathway blockade on viral immunity from immunopathology. Our findings complement those of Project 2 showing that ITIM and ITSM mutant mice have distinct outcomes in cancer and autoimmunity models, and Project 3 showing distinct outcomes in transplantation. Based on these data, we hypothesize that the PD-1 ITIM and ITSM motifs have distinct and overlapping functions, and that cell type and disease context will dictate which signaling motif is critical for controlling different aspects of the PD-1 inhibitory signal. In addition, we have discovered that the inhibitory receptors CD101 and CD112R are highly expressed on more terminally exhausted T cells with distinctive functional properties during chronic viral infection. Projects 2 and 3 have identified distinctive Tregs that highly express CD112R and CD101. Our findings lead us to hypothesize that CD101 and CD112R may serve as additional brakes on CD8 T cells in chronic infection, reducing their ability to respond to PD-1 blockade. To test these hypotheses, our Specific Aims are to 1) investigate roles of the PD-1 ITIM and ITSM motifs in controlling protective immunity versus immunopathology during acute and chronic viral infection; and 2) identify roles of CD101 and CD112R in controlling T cell exhaustion. Our goals are to elucidate mechanisms by which PD-1 regulates T cell exhaustion, identify new strategies for combination therapies to enhance T cell immunity during chronic viral infection, as well as new strategies to augment protective immunity during acute infections.